Multi-Layer Neuronal Imaging with Reverberation Multiphoton Microscopy

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ABSTRACT Many brain areas, such as neocortex and olfactory bulb, are vertically organized into layers containing distinct cell types that show different activity profiles and project to different downstream targets. Fast, volumetric imaging is thus indispensable to capture the dynamics of such neuronal populations within their stratified environments. While multiphoton microscopy (MPM) has become the gold standard for high resolution imaging from deep within brain tissue, it is generally restricted to 2D planar imaging. We propose to develop a technique to perform volumetric MPM where a long-range z-stack is acquired by near-instantaneous axial scanning, while maintaining 3D micron-scale resolution. Our technique, called reverberation MPM, enables the monitoring of neuronal populations over large scales, including the depth scale, with no speed penalty compared to conventional MPM. Reverberation MPM is a new technique which we have demonstrated only recently with proof of principle two-photon experiments. Much of our proposal will be focused on further developing this tool and characterizing its performance. Moreover, we propose to extend our technique to three-photon microscopy, for increased depth penetration. Our goal is to perform comprehensive 3D-resolved imaging of neuronal populations within volumes up to 1×1×1mm3, spanning the entire thickness of the mouse cortex. A key advantage of reverberation MPM is its extreme simplicity. It requires only the addition of a reverberation loop to a conventional MPM equipped with fast detection electronics. Moreover, it allows the acquisition of an arbitrary number of planes without increasing setup complexity. Other advantages are that our system is light efficient and easily compatible with video-rate scanning, making it ideal for volumetric calcium imaging using genetically encoded calcium indicators. These advantages make reverberation MPM particularly attractive as a general tool for fast, high resolution, large-scale volumetric imaging in brain tissue.